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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2017 Sep 11;98(4):191–202. doi: 10.1111/iep.12236

Influence of indigenous microbiota on experimental toxoplasmosis in conventional and germ‐free mice

Bruna B Nascimento 1, Christiane T Cartelle 1, Maria de L Noviello 1, Breno V Pinheiro 2, Ricardo W de Almeida Vitor 2, Danielle da G Souza 3, Simone de Vasconcelos Generoso 4, Valbert N Cardoso 4, Flaviano dos S Martins 3, Jacques R Nicoli 3, Rosa M E Arantes 1,✉
PMCID: PMC5639263  PMID: 28895246

Summary

Toxoplasmosis represents one of the most common zoonoses worldwide. Its agent, Toxoplasma gondii, causes a severe innate pro‐inflammatory response. The indigenous intestinal microbiota promotes host animal homoeostasis and may protect the host against pathogens. Germ‐free (GF) animals provide an important tool for the study of interactions between host and microbiota. In this study, we assessed the role of indigenous microorganisms in disease development utilizing a murine toxoplasmosis model, which includes conventional (CV) and GF NIH Swiss mice. CV and GF mice orally inoculated with T. gondii had similar survival curves. However, disease developed differently in the two animal groups. In CV mice, intestinal permeability increased and levels of intestinal pro‐inflammatory cytokines were altered. In GF animals, there were discrete epithelial degenerative changes and mucosal oedema, but the liver and lungs displayed significant lesions. We conclude that, despite similar survival curves, CV animals succumb to an exaggerated inflammatory response, whereas GF mice fail to produce an adequate systemic response.

Keywords: germ‐free mice, gut inflammation, microbiota, Toxoplasma gondii, toxoplasmosis

Toxoplasma gondii has a broad worldwide distribution, and approximately one‐third of the human population is serum positive for the parasite (Tenter et al. 2000). The acute phase of infection usually goes undetected, and human hosts may go through life with cysts in brain and muscle tissues. Infection occurs when an individual ingests raw or undercooked meat containing cysts, or water and foods contaminated with oocysts that release bradyzoites or sporozoites in the small intestine.

Once in the intestine, T. gondii rapidly invades the intestinal mucosa, where it becomes a tachyzoite capable of infecting mucosal and submucosal cells. Infected cells secrete cytotoxic molecules that attract neutrophils, macrophages, monocytes and T cells (Mennechet et al. 2004). Immune cells, in turn, secrete cytokines that stimulate the adaptive immune response. Dendritic cells capture and process parasite antigens for presentation and stimulation of T cells. CD4+ T‐lymphocytes in the lamina propria, in synergy with infected enterocytes, secrete pro‐inflammatory cytokines such as TNF‐α and IFN‐γ. The latter induces macrophages, dendritic cells and enterocytes to produce microbicidal molecules such as NO, which should eliminate the parasite (Buzoni‐Gatel et al. 2006). Comparisons of germ‐free (GF) and conventional (CV) animals have confirmed a role for the indigenous microbes in inflammatory and immune response.

Intestinal microbiota plays an important role in host animal homoeostasis (Sartor 2010; Martins et al. 2013; Sanders et al. 2013) and is vital to many aspects of normal host physiology. They are also related to progression of many diseases, especially the enteric infections. Disruptions of the microbial community changes host susceptibility to infection, and the infection itself can disturb the microbiota and the host inflammatory response (Sekirov & Finlay 2009; Sanders et al. 2013). Currently available data comparing GF and CV animals indicate that the indigenous microbiota almost always has a profound influence on host–parasite relationships during bacterial and protozoan infections (Sartor 2010). Sometimes the presence of the indigenous microbiota is essential for the pathogenicity of some protozoa and helminths (Phillips & Wolfe 1959; Vieira et al. 1987; Torres et al. 2000). In contrast, the microbiota can reduce the pathological consequences of other infectious diseases, as described for experimental infections with protozoa, fungi and helminthes (Salkowski et al. 1987; Silva et al. 1987; Martins et al. 2000) and almost all enteropathogenic bacteria (Wilson 1995).

In the current study, we assessed the potential role of the indigenous microbiota on the development and outcome of toxoplasmosis by comparing the clinical, histopathological and immunological parameters of CV vs. GF mice that were orally inoculated with T. gondii. Proposed mechanisms of probiosis include alterations of composition and function of the human gut microbiome, and corresponding effects on immunity and neurobiology. Intestinal microbes can alter gene expression in the mammalian gut mucosa, ultimately affecting the function of the gastrointestinal tract. A study using GF and CV mice revealed that the gut microbiota modulated the expression of many genes in the human or mouse intestinal tract, including genes involved in immunity, nutrient absorption, energy metabolism and intestinal barrier function (Larsson et al. 2012). Interestingly, most changes occurred in the mucosa of the small intestine. The presence of probiotics in the gastrointestinal tract can also affect patterns of gene expression, as demonstrated in a recent human study (Van Baarlen et al. 2010). Host–microbiota interactions were explored in the context of gut barrier function, pathogenic bacteria recognition and the ability of the immune system to induce either tolerogenic or inflammatory responses. There was speculation that the gut microbiota should be considered a separate organ, and whether analysis of an individual's microbiota could be useful in identifying their disease risk and/or therapy. However, more research is needed into specific diseases, different population groups and microbial interventions including probiotics. Based on our results, we suggest that uncovered mechanisms underlying the host–T. gondii–microbiota interaction in this relevant human disease should be further addressed to promote effective strategies, such as prophylactic and therapeutic probiotics use.

Methods

Animals

Germ‐free and CV 6‐ to 8‐week‐old female NIH Swiss mice (Taconic, Germantown, NY, USA) were used in this study. Water and commercial autoclavable diets (Nuvital, Curitiba, PR, Brazil) were sterilized by steam and administered ad libitum. Germ‐free mice were housed in flexible plastic isolators (Standard Safety Equipment Company, McHenry, IL, USA) and handled according to established procedures. Experiments with gnotobiotic mice were carried out in micro‐isolators (Uno Roestvaststaal, BV, Zevenaar, The Netherlands). Conventional animals were obtained from the animal breeding facility [Centro de Bioterismo (CEBIO)] of the Universidade Federal de Minas Gerais (UFMG). Germ‐free and CV mice were maintained in a ventilated animal caging system (Alesco Ltda., Campinas, SP, Brazil) with controlled lighting (12‐h light–dark cycle), humidity (60–80%) and temperature (22 ± 1°C) and were monitored daily.

Experimental infection

Cysts were obtained from the brains of Swiss mice that were chronically infected with the intermediate virulence strain TgCTBr07, genotype 67 described elsewhere (Carneiro et al. 2013). Brains were macerated in haemolysis tubes, after which 1 ml of sterile PBS was added and the solution was homogenized. Cysts were counted under light microscopy, and the final concentration was adjusted to 10 cysts per 0.1 ml of sterile PBS. Animals were then inoculated by oral gavage, and, in 30 days, cysts were available for either strain maintenance or experimental use (Carneiro et al. 2013; Pinheiro et al. 2015).

Experimental design

Survival was determined by daily inspection postinfection (p.i.) until day 14, and mice were weighed on alternate days to monitor the systemic repercussions during the course of infection.

Intestinal permeability analysis involved 12 CV and 12 GF animals divided into four groups of six individuals: control CV (CTL‐CV), infected CV (TgCTBr07‐CV), control GF (CTL‐GF) and infected GF (TgCTBr07‐GF). Histological and immunological analyses involved 20 CV and 20 GF animals grouped as above, with 10 individuals per group. At the end of the experimental period (day 8 of infection), all animals were bled from the axillary plexus under xylazine/ketamine anaesthesia – xylazine (15 mg/ml; Schering‐Plough Coopers, Cotia, SP, Brazil) and ketamine (80 mg/ml; Syntec Brasil Ltda, Cotia, SP, Brazil) by the i.p. route (Araujo et al. 2013). Following this procedure, animals were euthanized by cervical dislocation, and the small intestine, liver, spleen, lungs and brain were collected for further analysis, as well as the intestinal fluid for the assessment of secretory immunoglobulin A (sIgA) levels.

Intestinal permeability

Intestinal permeability was determined by measuring radioactivity diffusion in the blood after intragastric administration of diethylenetriamine pentaacetic acid (DTPA) labelled with 18.5 megabequerel (MBq) of 99 m‐technetium (DTPA‐99mTc). Diethylenetriamine pentaacetic acid is a macromolecule that rarely crosses the gastrointestinal barrier. However, when intestinal permeability is increased as a result of damage to the mucosa, DTPA permeation through the intestine (paracellular pathway) is permitted, and greater concentrations of this macromolecule can be measured in the bloodstream (Jørgensen et al. 2006; Costa et al. 2014). After eight days of infection, all mice received 0.1 ml of DTPA‐ 99mTc solution by gavage. Then, after 240 min, all animals (six per group) were anesthetized as described (Araujo et al. 2013) and 500 μl of the blood was collected and placed in appropriate tubes for radioactivity quantification. The data were expressed as % dose, using the following equation: % Dose = (cpm of blood/cpm of administered dose) × 100, where cpm represents counts of radioactivity per minute (Viana et al. 2010).

Intestinal sIgA levels

After euthanasia, the small intestines of mice from all groups were removed, and the contents were withdrawn, weighed and suspended in PBS using 500 mg of intestinal content per 2.0 ml PBS supplemented with an anti‐protease cocktail. After centrifugation at 2000 g and 4°C for 30 min, the supernatant was collected and kept frozen at −80°C until use. Immunoglobulin levels in intestinal fluid were evaluated by ELISA using goat anti‐mouse IgA (Sigma Chemical Co., St. Louis, MO, USA) and horseradish peroxidase‐conjugated goat anti‐mouse IgA (A‐4789; Sigma). Colour was developed with o‐phenylene‐diamine (Sigma), and absorbance at 492 nm was determined with an ELISA plate reader. The immunoglobulin concentrations were determined using a purified mouse IgA standard (Southern Biotechnology Associates Inc., Birmingham, AL, USA; Rodrigues et al. 2000; Martins et al. 2009).

Myeloperoxidase (MPO) activity assay

The extent of neutrophil accumulation in the tissue (intestine, liver, spleen and lung) was measured by assaying myeloperoxidase activity (MPO), as previously described. Briefly, the tissue (1 g of tissue per 1.9 ml of buffer) was homogenized in pH 4.7 buffer (0.1 mol/l NaCl, 0.02 mol/l NaPO4, 0.015 mol/l Na‐ethylenediaminetetraacetic acid) and centrifuged at 10,621 g (Eppendorf 5430 R, São Paulo, SP, Brazil) for 10 min, and the pellet was subjected to hypotonic lyses (1.5 ml of 0.2% NaCl solution followed 30 s later by the addition of an equal volume of a solution containing 1.6% NaCl and 5% glucose). Next, the pellet was resuspended in 0.05 mol/l NaPO4 buffer (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide and re‐homogenized. One millilitre aliquots of the suspension were transferred into 1.5‐ml Eppendorf tubes (Eppendorf 5430 R) followed by three freeze–thaw cycles using liquid nitrogen. The aliquots were then centrifuged for 15 min at 10,621 g, the pellet was resuspended in 1 ml, and samples were diluted before assay. Myeloperoxidase activity in the resuspended pellet was assayed by measuring the change in optical density (OD) at 450 nm using tetramethylbenzidine (1.6 mmol/l) and H2O2 (0.5 mmol/l). Results were expressed as the total number of neutrophils by comparing the OD of tissue supernatant with the OD of murine peritoneal neutrophils processed in the same way. Neutrophils were isolated from the peritoneal cavity of mice injected with 3 ml of casein 5%. A standard curve of neutrophil (95% purity) numbers vs. OD was obtained by processing purified neutrophils as described above and assaying for MPO activity (Souza et al. 2002).

N‐acetylglucosaminidase (NAG) activity assay

As a surrogate measure of macrophage activity, NAG activity was determined by enzymatic testing with the substrate p‐nitrophenyl‐N‐acetyl‐β‐d‐glucosamine (Sigma‐Aldrich, St. Louis, MO, USA). Tissue fragments were weighed and re‐suspended at a ratio of 100 mg of tissue for 1.0 ml of 0.9% saline solution at 4°C containing 0.1% v/v Triton X‐100 (Merck, Darmstadt, Germany). The homogenized solutions were centrifuged at 10,621 g, for 10 min, at 4°C. Supernatants were then immediately collected and used for the NAG assay, performed according to the manufacturer's instructions. Results were expressed in relative units (Bailey 1988).

Intestinal cytokine and chemokine levels

The concentration of TNF‐α, IFN‐γ, RANTES/CCL5, IL‐10 and TGF‐β in samples was measured in the intestine using commercially available antibodies and according to the procedures supplied by the manufacturer (R&D Systems, Minneapolis, MN, USA). These samples were weighed and homogenized in 1 ml of PBS (0.4 M NaCl and 10 mM NaPO4) containing anti‐proteases (0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA and 20 KI aprotinin A) and 0.05% Tween‐20. The samples were then centrifuged for 10 min at 12,000 g, and the supernatant was immediately used for ELISAs at a 1:3 dilution in PBS, as previously described (Souza et al. 2004; Amaral et al. 2008).

Histopathological analysis

Intestines were stretched on filter paper and opened from the anti‐mesenteric border. Intestinal contents were cleared without damage to the mucosal layer. Tissue samples were then prefixated for 10 min in a container with Bouin's solution with 2% glacial acetic acid. The prefixated intestines were placed on a flat surface and rolled from the distal to the proximal side with the mucosal layer facing inwards (Calvert et al. 1989; Arantes & Nogueira 1997). The intestinal rolls and other organs (liver, spleen, lung and brain) were then fixated by immersion in a 4% formaldehyde solution for 24 h. Samples were routinely processed, embedded in paraffin and submitted to microtome, so that 4‐μm‐thick histological pieces were obtained. Slides were stained with haematoxylin and eosin (H&E) for histological analysis.

Morphometric analysis

Tissue images were captured with a Cool SNAP‐Proof Color video camera (Media Cybernetics, Bethesda, MD, USA) and transferred to the computer using the image‐pro express software version 4.0 for Windows (Media Cybernetics). Blinded measurements were taken using the imagej software (version 1.47f; Wayne Rasband/National Institutes of Health, USA – available online at https://imagej.nih.gov/ij/).

For small intestine analyses, 10 photomicrographs of H&E‐stained slides were obtained with the 4× objective lens of an Olympus BX51 microscope (Olympus, Tokyo, Japan). The extent of intestinal lesion was defined as the ratio between the total perimeter of affected areas and the total perimeter of the organ. Affected areas were those presenting lesion signs defined by the loss of tissue architecture, superficial erosion and epithelial inflammation. For the lung morphometric analyses, the 10× objective lens was used and results were expressed as the ratio between septal and total areas (da Rodrigues‐Machado et al. 2010; Muniz et al. 2015).

Semi‐quantitative liver analysis

Blinded liver samples stained with H&E were analysed under light microscopy. A histopathological score was given according to the following metric: 0 – no alterations to the parenchyma and no increase in cellularity; 1 – A maximum of one isolated focus of inflammatory mononuclear cells (<10 cells) in all fields; 2 – Up to two infiltrated foci per field distributed in the parenchyma (>10 cells per focus); 3 – Frequent foci, two to six per field; 4 – Frequent and convalescent foci (>5 per field) accompanied by necrosis. All samples from two repeated experiments were examined with the 10× objective lens, n = 5.

Statistical analysis

Statistical significance was determined for a minimum of two independent experiments using analysis of variance (ANOVA) followed by Newman–Keuls or Bonferroni tests. The survival curve was analysed with the log‐rank survival test. graphpad prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA) was used for graph development and statistical analyses. Differences were considered statistically significant when P ≤ 0.05.

Ethical approval statement

All of the animal experiments were approved based on the regulations and guidelines of the Ethical and Animal Use Committee on Animal Experimentation (CEUA/UFMG protocol n° 394/2013).

Results

Survival rates did not differ between CV and GF animals infected with T. gondii

Survival rates did not differ significantly between T. gondii‐infected CV (TgCTBr07‐CV) mice and infected GF (TgCTBr07‐GF) mice (P = 0.2417; Figure 1a). At day seven of infection, CV animals had lost more weight than GF animals (P < 0.001). This difference, however, was no longer present on day eight (Figure 1b). The animals did not present signs of pain or distress by clinical observation, except for temporary and discrete loss of weight around day 7. At day 8, the animals were euthanized.

Figure 1.

Figure 1

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Survival Assessment (a), ponderal indexes (b), and sIgA levels (c) of CV and GF mice orally infected with T. gondii (TgCTBr07). Survival curves were analysed with log‐rank survival, n = 10; ponderal indexes were submitted to two‐way anova followed by Bonferroni tests, n = 10; sIgA levels were submitted to a one‐way anova test, followed by the Newman–Keuls test, n = 6; ***P < 0.001. Results were expressed as mean ± SD. CTL, control; TgCTBr07, Toxoplasma gondii strain; CV, conventional mice; GF, germ‐free mice.

T. gondii infection increased sIgA production and intestinal permeability in CV but not GF mice

Control CV (CTL‐CV) animals produced more sIgA than CTL‐GF mice (Figure 1c). Moreover, infection significantly increased sIgA production in CV but not in GF mice (Figure 1c). CTL‐CV and CTL‐GF mice had similar intestinal permeability. However, infection resulted in greatly increased permeability in CV (TgCTBr07‐CV) but not GF (TgCTBr07‐GF) animals (Figure 2e).

Figure 2.

Figure 2

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Intestinal pathology eight days postinfection in CV and GF animals. (a–d) Photomicrographs of H&E‐stained small intestine sections from Swiss mice are representative of four animals per group. (a) Control CV mice and (c) control GF mice; (b) infected CV mice with severe lesions in the mucous layer, oedema on top of villi (arrow) and loss of epithelial integrity (*); (d) infected GF mice with mild ileitis. Bar = 100 μm. (e) Intestinal permeability in CV and GF animals infected with the TgCTBr07 strain of T. gondii (n = 5) expressed as % dose in blood. (f) Extension of injury expressed as ratio of lesion perimeter to total organ perimeters (n = 4). The entire extension of the intestine was analysed with a 4× objective lens in H&E‐stained slides. (g) MPO and (h) NAG activity expressed as units/mg of intestine (n = 6). One‐way anova test, followed by Newman–Keuls test; *P < 0.05; **P < 0.01; ***P < 0.001. Results were expressed as mean ± SD. CTL, control; TgCTBr07, Toxoplasma gondii strain; CV, conventional mice; GF, germ‐free mice. [Colour figure can be viewed at wileyonlinelibrary.com].

T. gondii‐induced lesions in the small intestine were larger in CV than in GF mice

The ileum and jejunum of TgCTBr07‐CV mice displayed histopathological alterations with similar distribution and intensity. There was focal loss of integrity in the mucosal layer, considerable height variation of the jejunal villi, increased cellularity in the mucosal and submucosal layers, and oedema on the top of villi associated with loss of the brush border. There were areas of epithelial cell degeneration, especially on the surface of villi (Figure 2b).

The ileum and jejunum of TgCTBr07‐GF mice also displayed alterations with similar distribution and intensity, albeit to much lesser degrees than those observed in TgCTBr07‐CV mice. The mucosal layer in GF mice was mostly preserved, and only subtle epithelial degeneration was observed (Figure 2d). Moreover, morphometric analysis showed that lesions were larger in TgCTBr07‐CV compared to both control groups and TgCTBr07‐GF mice (Figure 2f).

The presence of neutrophils and macrophages was assessed through the activity of specific enzymes in the small intestine: MPO (Figure 2g) and NAG (Figure 2h) respectively. The TgCTBr07‐CV mice had decreased MPO activity when compared to controls and TgCTBr07‐GF mice. There were no significant differences in NAG activity among groups.

Regarding TNF‐α (Figure 3a), levels were similar between the two control groups and were elevated in TgCTBr07‐CV, but not TgCTBr07‐GF mice. Levels of IFN‐γ (Figure 3b) were elevated by infection in CV and GF mice but more pronounced in the latter. Levels of chemokine RANTES/CCL5 (Figure 3c) were elevated by infection in CV and GF mice but more pronounced in the former. Finally, levels of anti‐inflammatory cytokines IL‐10 (Figure 3d) and TGF‐β (Figure 3e) were not significantly different among groups.

Figure 3.

Figure 3

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Ileum cytokine and chemokine levels in CV and GF mice: TNF‐α (a), IFN‐γ (b), RANTES/CCL5 (c), IL‐10 (d), TGF‐β (e). CTL, control; TgCTBr07, Toxoplasma gondii strain; CV, conventional mice; GF, germ‐free mice. One‐way anova test, followed by Newman–Keuls test; *P < 0.05; **P < 0.01; ***P < 0.001. Results were expressed as mean ± SD.

More neutrophils and macrophages were recruited by the liver in TgCTBr07‐CV than in TgCTBr07‐GF mice

Livers of TgCTBr07‐CV mice displayed numerous diffuse inflammatory foci mixing polymorphonuclear and mononuclear cells in the hepatic parenchyma along with intense vacuolar alterations to hepatocytes (Figure 4b). On the other hand, livers of TgCTBr07‐GF mice were better preserved with sparse foci of smaller volume and subtle degeneration of hepatocytes (Figure 4d). Quantitatively, the histopathology score of hepatic lesions was also significantly lower in TgCTBr07‐GF compared to TgCTBr07‐CV mice (Figure 4e). These results were aligned with the analyses of MPO and NAG activities in the liver (Figure 4f and 4g respectively).

Figure 4.

Figure 4

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Liver pathology 8 days postinfection in CV and GF animals. (a–d) Photomicrographs of H&E‐stained liver sections from Swiss mice are representative of four animals per group. (a) Control CV mice and (c) control GF mice; (b) infected CV mice with a larger number of inflammatory foci (arrows) than (d) infected GF mice. Bar = 100 μm (n = 4). (e) Liver histological score of CV and GF mice, obtained as described in Methods (n = 5). (f) MPO and (g) NAG activity expressed as units/mg of liver (n = 6). One‐way anova test, followed by Newman–Keuls test; **P < 0.01; ***P < 0.001. Results were expressed as mean ± SD. CTL, control; TgCTBr07, Toxoplasma gondii strain; CV, conventional mice; GF, germ‐free mice. [Colour figure can be viewed at wileyonlinelibrary.com].

More neutrophils and macrophages are recruited by the spleen in TgCTBr07‐CV than in TgCTBr07‐GF mice

CV (Figure 5a) and GF (Figure 5c) mice presented similar spleen histopathology except for a slight decrease of the follicles and red pulp cellularity; that is, infection induced an increase in the cellularity of germinal centres and red pulp cellularity in CV animals (Figure 5b), with almost no alteration in GF animals (Figure 5d).

Figure 5.

Figure 5

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Spleen pathology 8 days postinfection in CV and GF animals. Photomicrographs of H&E‐stained spleen sections from Swiss mice are representative of four animals per group. (a) Control CV mice and (c) control GF mice; (b) infected CV mice and (d) infected GF mice. Notice the decrease in follicles and red pulp cellularity in GF animals; infected CV has increased cellularity of germinal centres. Bars = 100 μm (n = 4). (e) MPO and (f) NAG activity expressed as units/mg of spleen (n = 6). Results were expressed as mean ± SD. CTL, control; TgCTBr07, Toxoplasma gondii strain; CV, conventional mice; GF, germ‐free mice. One‐way anova test, followed by Newman–Keuls test; *P < 0.05; **P < 0.01; ***P < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com].

The assessment of neutrophil and macrophage spleen populations through MPO and NAG activities respectively (Figure 5e,f), showed that TgCTBr07‐CV mice recruited more immune cells to the organ than TgCTBr07‐GF animals.

Pulmonary lesions are similar in TgCTBr07‐CV and TgCTBr07‐GF mice

The ratio of septal tissue area to total tissue area was used as a measure of lung lesion. In CTL‐CV and CTL‐GF animals, ratios were 54% and 44% respectively (Figure 6e). CTL‐GF, when compared to CTL‐CV mice, had noticeably thinner septal tissue with fewer cells (Figure 6a,c). T. gondii infection elevated the ratio of septal tissue in CV mice (Figure 6b) and slightly higher in GF animals (Figure 6d,e). In GF animals, the reduction in air space largely resulted from atelectasis, whereas in TgCTBr07‐CV mice, lung MPO activity increased significantly and septal tissue grew with infiltrated neutrophils (Figures 6b,d,f).

Figure 6.

Figure 6

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Lung pathology 8 days postinfection in CV and GF animals. Photomicrographs of H&E‐stained lung sections from Swiss mice are representative of four animals per group. (a) Control CV mice and (c) control GF mice; (b) infected CV mice and (d) infected GF mice. Arrows indicate enlarged alveolar septal areas, and * indicates increased cellularity. Bars = 100 μm. (e) Morphometric analysis of pulmonary lesions – ratio of septal to total area – obtained from 10 fields with a 10× objective lens from H&E‐stained slides, n = 4. (f) MPO (n = 6). Results were expressed as mean ± SD. CTL, control; TgCTBr07, Toxoplasma gondii strain; CV, conventional mice; GF, germ‐free mice. One‐way anova test, followed by Newman–Keuls test; *P < 0.05; **P < 0.01; ***P < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com].

TgCTBr07‐GF mice are protected against early meningitis

Eight days after infection, TgCTBr07‐CV mice displayed moderate alterations of the meninges and increased cerebral vascularity, whereas no changes were detectable in TgCTBr07‐GF mice (Figure 7b,d).

Figure 7.

Figure 7

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Brain pathology 8 days postinfection in CV and GF animals. Photomicrographs of H&E‐stained brain sections from Swiss mice are representative four animals. (a) Control CV mice and (c) control GF mice; (b) infected CV mice and (d) infected GF mice. Arrow indicates oedema, and *indicates increased cellularity in the meninges of CV animals. Bar = 100 μm. [Colour figure can be viewed at wileyonlinelibrary.com].

Discussion

Previous studies have shown that C57BL/6 mice orally infected with T. gondii developed intestinal histological abnormalities (Liesenfeld 2002; Pinheiro et al. 2015). In the current work, we utilized Swiss mice, allogeneic animals that develop similar intestinal problems, which allow us to compare our results with those of other studies (Liesenfeld 2002). One group of us (RWV) recently genotyped 25 of 27 T. gondii samples obtained from the peripheral blood of newborn infants with congenital toxoplasmosis using PCR‐RFLP. Here, we used one of the isolated strains with intermediate virulence (TgCTBr07), which kills animals within the initial two weeks after infection, not allowing lesions to become chronic (Carneiro et al. 2013).

T. gondii orally infects the host, quickly inducing a cascade of immunological events that involves innate and adaptive responses. In C57BL/6 mice, the parasite also induces severe intestinal inflammation, characterized by loss of epithelial architecture, shortened villi, and massive influx of inflammatory cells in the lamina propria and necrotic plaques (Liesenfeld et al. 2001), much like we observed here in infected CV animals.

Considering the route of entry of T. gondii through the intestine, we hypothesized that the cell interactions that regulate responses to antigens of the normal bacterial microbiota in the presence of an intact epithelial barrier could be affected in GF conditions. However, survival curves were similar for infected CV and GF mice, and most animals from both groups died between days 11 and 14 after inoculation, when only TgCTBr07‐CV mice developed clinical signs of T. gondii infection that have previously been described in mice, such as erectile body hair, postural arching and excessive motor activity. Moreover, CV animals experienced lower weight gain compared to GF animals starting between days 6 and 8 after infection when they were euthanized in our experimental groups. The relatively better clinical status of GF mice might be related to the reported heightened pain threshold of these animals (Amaral et al. 2008). Also, it could be possible that the differences in phenotypes were due to differences in infection efficiencies, but we did not determine the parasitic loads. In fact, the influence of microbiota on the course of an infectious disease is unpredictable and the operator’s mechanisms for protection or susceptibility have received attention in GF and CV mice infected with bacterial, enteropathogenic bacteria, protozoan, helminths and fungi (Sartor 2010), bacteria (Phillips & Wolfe 1959; Salkowski et al. 1987; Silva et al. 1987; Vieira et al. 1987; Wilson 1995; Martins et al. 2000; Torres 2000).

In the present study, TgCTBr07‐CV mice had larger lesions and more severe ileitis compared to TgCTBr07‐GF mice despite similar survival curves. Previous studies showed that T. gondii‐induced ileitis worsens during the first eight days postinfection, from mild inflammation (3–5 days) to severe necrosis (8 days). This development parallels a rise in commensal Gram‐negative gut bacteria identified as Escherichia coli and Bacteroides/Prevotella, accompanied by a pronounced loss of bacterial diversity (Heimesaat et al. 2006).

We also showed increased intestinal permeability in TgCTBr07‐CV mice compared to TgCTBr07‐GF mice. TgCTBr07‐CV showed more lesions of intestinal epithelium, consistent with their higher intestinal permeability. The intestinal barrier, among many other functions, provides a filter that separates potentially harmful luminal contents from the internal environment of the body. Thus, any harm to this barrier might contribute to the translocation of luminal contents, such as bacteria and toxins, to the portal and systemic circulation (MacFie 2004; Gatt et al. 2007; Yan et al. 2009; Tran et al. 2015). As described in other models of infection and intestinal damage, the GF animals do not present significant increase in intestinal permeability while CV animals usually do (Canesso et al. 2014; Pedroso et al. 2015). That behaviour has been attributed to the integrity of GF barrier in the absence of inflammatory cells and microbiota.

Polymeric sIgA participates in the humoral defence mechanism, ensuring the barrier function of mucosal surfaces by preventing both the adherence of bacteria to mucosal surfaces and the penetration of antigens into the organism's internal environment (Holmgren & Czerkinsky 2005; Brandtzaeg 2010). The intestinal microbiota promotes the activation of plasma cells that produce and secrete sIgA. Our results are aligned with previous work showing a more pronounced sIgA production in CV than in GF animals (Moreau et al. 1978; Pabst 2012). Nevertheless, elevated sIgA was not sufficient to counter the effects of T. gondii on the intestinal wall, nor did it stop bacterial and pathogen translocation through the intestinal epithelium, contrary to others studies (Duerkop et al. 2009; Kelly & Mulder 2012).

T. gondii‐induced ileitis results from a Th‐1‐type immunopathology characterized by T cell‐mediated elevation of pro‐inflammatory mediators including IFN‐γ, TNF‐α and NO (Heimesaat et al. 2006). We reproduced the profile of cytokines associated with T. gondii‐infected CV mice. The IL‐12 cytokine is crucial for the activation of natural killer (NK) cells that produce IFN‐γ (Reis e Sousa et al. 1997). Natural killer cells have several chemokine receptors that facilitate this process, among them CCR5. Reduced NK cell migration to the infection site has been reported for CCR5−/− mice inoculated with T. gondii. This impairment results in a lower inflammatory response to infection, and infected CCR5−/− mice eventually die of parasitaemia (Khan et al. 2006). In the current study, TNF‐α and RANTES/CCL5 levels were significantly elevated in the intestines of infected CV animals in comparison with control and infected GF animals. Our results show a correlation between the severity of ileitis in CV mice and the infection‐induced TNF‐α level. Nevertheless, both CV and GF animals succumbed to infection after similar periods.

A significant increase in IFN‐γ was observed in infected animals, both CV and GF. However, GF animals produced at least twice as much IFN‐γ as CV mice. Blocking IFN‐γ, TNF‐α and NO activity prevents ileum necrosis and mouse mortality, demonstrating that inflammation, and not direct parasite action, causes lesions (Liesenfeld et al. 1996, 1999). However, in the current study, elevated production of IFN‐γ by infected GF mice did not correlate with increased pathology or mortality.

An adequate pro‐inflammatory response provides essential protection against T. gondii infection; however, an exaggerated or uncontrolled response will often result in pathology. The host must balance the response in order to maximize pathogen elimination while minimizing damage to itself (Shaw et al. 2006; Rescigno & Di Sabatino 2009). Production of IL‐10 and TGF‐β leads to the activation of regulatory T cells that inhibit the immune response and induce mucosal tolerance (Barnes & Powrie 2009). The anti‐inflammatory cytokine IL‐10 attenuates host response to T. gondii. As previously shown, IL‐10−/− mice do not neutralize IFN‐γ and usually die during the acute phase of diseases (Gazzinelli et al. 1996). In the current study, we did not detect significant IL‐10 production in any of the experimental groups, a finding that may have resulted from the number of inoculated cysts (10) or from the short time that elapsed after infection until the time samples were collected (8 days).

Other anti‐inflammatory cytokines involved in immunopathology regulation include TGF‐β and IL‐27. Intestinal epithelial lymphocytes secrete TGF‐β, which acts on the control of intestinal pathology after oral infection with T. gondii (Mennechet et al. 2004). Despite the lack of significance, our GF‐ and CV‐infected animals showed higher levels of the cytokine when compared to their non‐infected controls. Also, the levels of IFN‐γ correlated inversely with the levels of TGF‐β in both groups.

We also assessed systemic differences between CV and GF animals by evaluating lesions to the liver, spleen, lungs and brain 8 days after infection. The liver is frequently targeted by tachyzoites that kill hepatocytes and Küpffer cells, creating necrotic foci and mononuclear cell infiltrates (Frenkel 1988). Infection was significantly less damaging to the livers of GF than CV mice (Figure 4b,d).

Additionally, inflammation markers MPO and NAG were also more elevated in infected CV livers. Thus, the presence of an indigenous microbiota was associated with increased intestinal permeability, bacterial translocation and with more severe hepatic lesions induced by the parasite.

The spleens of TgCTBr07‐CV mice, collected 8 days after infection, had reduced lymphoid follicle areas, atypical nodular structures and loss of the usual architecture. Death of humans and animals with toxoplasmosis is often preceded by lymphocyte depletion (Frenkel 1988). Levels of MPO and NAG were elevated in the spleens of infected CV mice compared to CTL‐CV animals, but not in the organs of infected GF animals.

The lungs of TgCTBr07‐CV mice had lesions characterized by increased septal cellularity caused by neutrophils, as evidenced by the elevated MPO activity. Both CV and GF mice had increased septal thickness and extensive areas with atelectasis quantified as a ratio of septal to total tissue. This sort of pulmonary pathology could have caused respiratory failure and, ultimately, death. In fact, other authors have attributed T. gondii lethality in mice to pulmonary lesions (Shaw et al. 2006; Barnes & Powrie 2009). Previous work showed that, after oral infection with T. gondii, parasites rapidly reach the lungs before other organs in the abdominal cavity are infected (Boyle et al. 2007).

TgCTBr07‐CV mice had subtle to moderate meningitis that was predominantly mononuclear and that was not seen in TgCTBr07‐GF mice. The absence of brain lesions in mice during the acute phase of toxoplasmosis has been previously reported (Liesenfeld et al. 1996; Mordue et al. 2001; Smiley et al. 2005; Djurković‐Djaković et al. 2006; Boyle et al. 2007). The brain may be the last organ to be affected by tachyzoites because of its enhanced immunological protection. The blood–brain barrier slows the influx of infected cells, inflammation mediators and antibodies into the brain, which has greater pathological relevance during the chronic phase of infection (Frenkel 1988; Dubey et al. 2012). The lack of gut microbiota has been associated with increased BBB permeability and altered expression of tight junction proteins (Braniste et al. 2014); however, our results suggest that the GF condition may protect against toxoplasma‐induced inflammation during the early period of infection, as the meninges were not affected.

Conclusion

Intestinal microbiota plays a significant role in the early progression of toxoplasmosis in the murine model of infection with the TgCTBr07 strain. Although TgCTBr07‐CV and TgCTBr07‐GF mice had similar survival curves, disease development differed between the two animal lines. A large increase in intestinal permeability and alterations of pro‐inflammatory cytokines were observed in CV but not GF mice. Infection also resulted in more severe lesions in the liver, lungs and brain of CV animals. Inflammation markers were also more elevated in the spleen, liver and lungs of CV mice. Paradoxically, an elevation of IFN‐γ was also seen in the intestines of GF mice, which had less severe ileitis. Thus, we suggest that infected CV animals succumb to an exacerbated response of the immune system, whereas infected GF mice fail to produce an adequate systemic response. Further ongoing investigation is needed to clarify the mechanisms involved in the course and presentation of the disease in the absence of microbiota.

Conflict of Interest

The authors have no conflict of interest to declare.

Funding source

This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil) Grant 458832/2014‐6 and Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG/Brazil) Grant PPM/2016. B.B. Nascimento received a MSc scholarship from CNPq/Brazil. C.T. Cartelle and M.L. Noviello were funded by PNPD/CAPES grant 2248/2011. F.S. Martins, J.R. Nicoli and R.M.E Arantes received a CNPq Research Fellowship.

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